Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase

Partial amino acid sequence of 80 kDa oxidized protein hydrolase (OPH), a s erine protease present in human erythrocyte cytosol (Fujino et al., J. Bioc hem. 124 (1998) 1077-1085) that is adherent to oxidized erythrocyte membran es and preferentially degrades oxidatively damaged proteins (Beppu et al., Biochim. Biophys. Acta 1196 (1994) 81-87; Fujino et al., Biochim. Biophys. Acta 1374 (1998) 47-55) was determined. The N-terminal amino acid of diisop ropyl fluorophosphate (DFP)-labeled OPH was suggested to be masked. Six pep tide fragments of OPH obtained by digestion of DFP-labeled OPH with lysyl e ndopeptidase were isolated by use of reverse-phase high-performance liquid chromatography, and the sequence of more than eight amino acids from the N- terminal position of each peptide was determined. Results of homology searc h of amino acid sequence of each peptide strongly suggested that the protei n was identical with human liver acylpeptide hydrolase (ACPH). OPH showed A CPH activity when N-acetyl-L-alanine p-nitroanilide and N-acetylmethionyl L -alanine were used as substrates, Glutathione S-transferase (GST)-tagged re combinant ACPH (rACPH) was prepared by use of baculovirus expression system as a 107-kDa protein from cDNA of human erythroleukemic cell line K-562, r ACPH reacted with anti-OPH antiserum from rabbit. rACPH showed OPH activity when hydrogen Deroxide-oxidized or glycated bovine serum albumin was used as substrates. As well as the enzyme activities of OPH, those of rACPH were inhibited by DFP. The results clearly demonstrate that ACPH, whose physiol ogical function has not yet been well characterized, can play an important role as OPH in destroying oxidatively damaged proteins in living cells. (C) 2000 Elsevier Science B.V. All rights reserved.